Hepatitis C virus (HCV) is remarkable at disrupting human immunity to establish chronic infection; it is a global health problem with limited treatment options and no available vaccine. The mechanisms by which the virus persists in the majority of infected liver by overcoming the host innate to adaptive immunity is currently unclear, partly due to our incomplete understanding of HCV-host interactions that lead to this immune disruption. We have previously demonstrated that chronic HCV infection leads to immunodysregulation mediated through up- regulation of negative immunomodulators, including programmed death-1 (PD-1), suppressor of cytokine signaling-1 (SOCS-1), and most recently, T cell immunoglobulin and mucin domain protein-3 (Tim-3). While Tim-3 has been shown to play a critical role in T cell exhaustion during chronic viral infections, its expression and function on innate immune cells in HCV persistence and antiviral responses remain unknown. We have recently discovered that Tim-3 plays a pivotal role in negative regulation of Toll-like receptor (TLR)-mediated innate immune responses, being up-regulated on monocytes/macrophages (M/M?) isolated from chronically HCV-infected individuals and healthy M/M??co-cultured with HCV-expressing hepatocytes. Importantly, blocking Tim-3 signaling restores the expression of IL-12, a key pro-inflammatory cytokine linking innate to adaptive immune responses. This novel observation suggests that the inability to clear virus in chronically HCV-infected hosts may be a function of a Tim-3-mediated impairment of innate immunity, with subsequent dysfunction of adaptive immune responses. We thus hypothesize that HCV-mediated Tim-3 up-regulation on M/M??plays a central role in innate immune and IL-12 dysregulation, such that blockade of Tim-3 signaling in M/M??may rescue impaired antiviral immune responses. To test this hypothesis, we will carry out the following specific aims: 1) Define the role of Tim-3 up-regulation on M/M? from HCV-infected patients following antiviral therapy with defined outcomes, comparing with those naturally resolved HCV infection or healthy subjects, by examining both Tim-3 expression and M/M??functions, and in particular, IL-12 production. 2) Determine the mechanisms by which Tim-3 is up-regulated on M/M??using an HCV-expressing hepatocyte model system, focusing on specific HCV antigen-mediated regulation of Tim-3 transcription, translation, biosynthesis and degradation. 3) Determine the effects of Tim-3 signaling in M/M??on host antiviral responses, including dendritic cell (DC) IL-12 expression, virus-specific CD4+ and CD8+ T lymphocyte responses, hepatocyte interferon (IFN) signaling and HCV replication. The overall goal of this proposal is to employ a translational approach to obtain a unified overview of how HCV-mediated Tim-3 up-regulation on M/M? alters IL-12 expression and host innate to adaptive immune responses to HCV infection, so as to develop effective strategies to combat this common viral disease. PUBLIC HEALTH RELEVANCE: HCV is characterized by persistent infection and poor treatment response. Why this virus is able to persist in the majority of infected individuals, and more than half of the most prevalent genotype 1 HCV-infected patients fail the standard antiviral treatment, remains unclear; but disruption of both innate and adaptive immune responses appears to play a major role. Based on our preliminary data, we suspect that a novel immunomodulator, Tim-3, is regulating the ability of innate immune cells, such as M/M?, to produce critical regulatory cytokines, such as IL-12, leading to disruption of subsequent T lymphocyte responses to HCV infection. This project will examine whether and how Tim-3 expression is regulated on M/M??in individuals with chronic HCV infection following antiviral therapy, with a focus on the virus and host interactions in a new system mimicking the HCV-infected liver as a model and manipulating Tim-3 signaling in M/M??as a tool. This study will facilitate effective approaches to improve viral clearance, and thus is significant and timely.